Optimization of germination and ultrasonic‐assisted extraction for the enhancement of γ‐aminobutyric acid in pumpkin seed

Abstract Germination and ultrasonic‐assisted extraction (UAE) are economical and effective methods to enhance bioactive compounds in plant seeds. We optimized the germination parameters and UAE parameters by using response surface methodology to maximize the recovery of γ‐aminobutyric acid (GABA) in pumpkin seeds. The optimal germination conditions were as follows: soaking the seeds at 28°C for 6 h with 0.2% CaCl2, 3.8 mg/ml monosodium glutamate, and 4.0 mg/ml vitamin B6, then germination at 30°C for 61.6 h. The optimal conditions for UAE were as follows: 1:75 (w/v) material‐to‐solvent ratio, 220 W ultrasonic power, and ultrasonic treatment at 50°C for 14.4 min, which afforded an extraction yield of 2679 ± 10 mg/100 g. Moreover, the GABA‐enhanced extract showed the potential of hypolipidemic effect in type 2 diabetes rats. These results confirmed that a combination of germination and UAE increased the GABA yield from pumpkin seeds and provided a basis for GABA‐enhanced production to improve lifestyle‐associated diseases.


| INTRODUC TI ON
Pumpkin is a commercially important crop and cultivated worldwide.
Pumpkin seeds are easily available and are rich sources of several functional components (Dotto & Chacha, 2020) including unsaturated oils, proteins, phytosterols, vitamin K, and γ-aminobutyric acid (GABA), which has beneficial effects on some lifestyle-associated diseases. The hypoglycemic and hypolipidemic effects of whole pumpkin seeds have been previously reported (Adams et al., 2011), promoting the acceptance of pumpkin seeds in the individuals with related lifestyle-associated diseases. Because of the nutritional and potential therapeutic values, interest in the application of pumpkin seeds has considerably increased in recent years. GABA (C 4 H 9 NO 2 ), a bioactive constituent of pumpkin seeds, is a 4-carbon nonprotein amino acid that plays a key role in living organisms (Bown & Shelp, 2016). GABA has many reported bioactivities, such as alleviation of anxiety, regulation of blood pressure and cholesterol, and prevention of obesity and diabetes (Chua et al., 2019;Imam et al., 2012;Sato et al., 2021;Shelp et al., 1999;Wu et al., 2021).
Therefore, investigation of GABA-enriched functional food is becoming increasingly important.
In plant, GABA is synthesized from the l-glutamic acid (Glu) in plants through catalytic activity of glutamate decarboxylase (GAD) (Bouché & Fromm, 2004). Thus, the concentration of Glu, the activity of GAD, and environmental factors contribute to the accumulation of GABA. Usually, naturally occurring GABA in plants is insufficient to meet the nutritional requirement as a functional food and chemically synthesized GABA is not permitted as a food additive (Wu & Shah, 2017). Accordingly, biochemical methods, such as fermentation, enzymatic hydrolysis, germination, and ultrasonic extraction, have been applied to enrich GABA in natural food production (Ding et al., 2016(Ding et al., , 2018Galli et al., 2022). Several literatures have demonstrated that germination is an economical and effective method for enriching bioactive compounds in plant seeds (Ding et al., 2016;Moongngarm & Saetung, 2010;Ohanenye et al., 2020). During the germination process, biosynthesis and interconversion reactions can result in the generation of new compounds and accumulation of metabolites (Kumar & Chauhan, 1993). Related studies showed that the germination treatment has a profound impact on the accumulation of functional components including GABA (Bhinder et al., 2022;Cáceres et al., 2014). Furthermore, temperature, pH, and other environmental factors can also impact the efficiency of GABA accumulation by altering the extent of germination (Cáceres et al., 2014;Zhang et al., 2014). Several studies have improved the nutritive value in pumpkin seeds through their germination (Quanhong & Caili, 2005).
However, there is still a lack of work on the optimization of parameters for the accumulation of GABA in pumpkin seed. Ultrasonicassisted extraction (UAE) is regarded as a green process for the extraction of valuable natural products (Naik et al., 2021). UAE has the advantages of high product yields with low maintenance costs and short processing times. Processes relying on UAE are affected by cavitation, thermal, and mechanical conditions. Bubbles formation and growth enhance the chemical activity in the solution when ultrasound transmits in liquid medium (Kiss et al., 2018), therefore, several probable mechanisms for UAE have been proposed. Based on the mechanical and cavitation effectiveness in UAE, the cell wall is disrupted and particle size is reduced by improving the mass transfer across the cell membrane, and the penetration, swelling, as well as hydration process are enhanced (Hossain et al., 2012;Vinatoru & Nenitzescu, 2001;Wang et al., 2008). UAE has been successfully applied to the extraction of functional compounds from grains and beans (Ding et al., 2018;Eze et al., 2022), but there is lack of studies on the optimization of UAE parameters applied in GABA extraction from pumpkin seed.
In this study, germination and UAE were combined to effectively improve the yield of GABA in pumpkin seeds. Response surface methodology (RSM) was applied to optimize the parameters in both the germination and UAE process. The impact of germination on GABA enrichment in pumpkin seed and the effect of UAE on GABA yield of the extraction were also established. Moreover, a rat model for T2DM was applied to reveal the bioactivity potential of the GABA extract from pumpkin seeds. This study aims to provide an economic, green, and efficient method for the production of GABAenriched functional food and reveal the potential of pumpkin seeds as a natural nutritional supplement in the daily diet.

| Experimental design and data analysis for optimization
Based on single-factor experiments and Plackett-Burman design, RSM was applied for the optimum germinant parameters and ultrasonic extraction parameters for high GABA content. Central composite design (CCD) was used to determine the combined effects of soaking temperature, MSG concentration, and germination time on GABA content. The experimental runs in CCD are shown in Table S1. Similarly, the four independent variables (solid-liquid ratio, ultrasonic power, time, and temperature) for UAE were run in a Box-Behnken design (BBD) to determine the optimum combinations for GABA yield. Detailed information of factors and levels are presented in Table S2.
The variation in GABA yield related to the independent variables was evaluated by a second-order polynomial regression model, as shown in Equation (2), which is a widely accepted empirical model in optimization process (Myer & Montgomery, 2002). According to Equation (2), linear, quadratic, and interactive effects of independent variables (X) on dependent variable (Y) were determined.
where Y denotes the response variable obtained from treatment combination of independent variables (X i , X i+1 , X j ), β 0 represents the intercept, and β i, β ii, and β ij are the linear, quadratic, and interaction regression coefficients of variables, respectively.

| GABA measurement
The content of GABA was calculated by using HPLC with precolumn PITC derivatization using a modified previously described method (Qingyun et al., 2008). Defatted sample (0.1 g) was mulled with 5 ml of distilled water for ultrasonic extraction, which lasted 20 min at 37℃, and then centrifuged. A 400 μl aliquot of the supernatant After holding for 1 h in darkness, 1.0 ml of n-hexane was added, and the mixture was shaken and on standing separated. The lower phase was collected and filtered through a 0.45 μm membrane filter. HPLC (LC-20A Shimadzu Co. Ltd) was applied to quantify GABA content. Samples (10 μl) were loaded onto a Venusil AA column  Table S3.

| Animal experiments
Animal experiment was carried out referring to the reported method (Liu et al., 2018) with some modification. Besides, all the experimental protocol was approved by the Ethics Committee of Beijing Laboratory Animal Research Center (SYXK2015-0046; Beijing, China). Adult male Wistar rats were obtained and kept in cages with SPF barrier environment (temperature of 21-23°C, relative humidity of 40%-45%, and 12 h light/dark cycle). All the rats had free access to food and water for 1 week in order to acclimatize. The rats received high-fat diet (HFD) (10% lard, 20% sucrose, 2.5% cholesterol, 1% sodium cholate, and 66.5% pulverized standard rat pellet) for 4 weeks for adaptation with the diet, and then the rats were injected with streptozocin (STZ) (30 mg/kg body weight) after adaptations.
The presence of polyuria and polydipsia along with fasting blood glucose higher than 11.1 mol/L indicated that the T2DM rat model was established successfully. The T2DM rats were randomly separated into three groups of 10 rats each: (1) control group (T2DM group), continually fed with HFD diet and intragastrically administered with (2) Metformin (MT) treatment group (MT group), fed with HFD diet and intragastrically administered with 200 mg/kg body weight metformin once daily; (3) GABA-enhanced extract from germinated pumpkin seeds (PSGE) treatment group (PSGE group), fed with HFD diet and intragastrically administered with 2 g/kg body weight of PSGE (GABA content is 25.0 ± 0.8 mg/g) once daily (Tian et al., 2011).
At the end of the experiment, blood samples were collected and promptly centrifuged to isolate the serum. Then, the serum was kept at a temperature of −60°C for biochemical analysis, including blood glucose (BG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL), and other blood biochemical parameters (Zhang et al., 2017).

| Selection of pumpkin seeds varieties for GABA accumulation
In present study, sprout length (SL), germination percentage (GP), GABA content, protein, and amino acid content were evaluated among seven cultivars after germinating for 72 h. The germination percentage and sprout length reflect the growth status of pumpkin seeds. As shown in Figure 1a,b, significant differences were observed among cultivars. YH-3 and XC-2 germinated effectively under the experimental condition, which resulted in germination percentage and sprout length of 89%, 40.0 mm and 97%, 47.0 mm, respectively. Figure 1c shows the changes in GABA content. For all the tested cultivars, the GABA content was significantly increased (p < .01) after germination. Before germination, RF-9 and YH-3 exhibited higher GABA content (653 mg/100 g, 622 mg/100 g), while the GABA content of other cultivars range from 310 mg/100 g to 488 mg/100 g. After germination for 72 h, GABA accumulated in all cultivars, but maximum GABA content (2150 mg/100 g) was found in XC-2. This was an increase of five times present in raw seeds. Based on GABA content after germination, the sequence was XC-2 > YH-3 > JH-4 > RF-9 > LR-2 > LR-3 > LR-1. It has been reported that soybeans with lower initial GABA content show higher GABA accumulation efficiency after germination (Huang et al., 2017). This is in agreement with our current study on the different pumpkin seeds cultivars.
Changes in total protein (TP) and amino acid (AA) content in pumpkin seeds were also affected by germination. The TP content of YH-3, LR-3, RF-9, XC-2, and LR-2 diminished after germination (Table 1). During germination, part of the protein in seeds is degraded, providing energy and nutrition for seeds to grow. TP of LR-1 and JH-4 increased but these differences were not statistically significant. Meanwhile, AA content of YH-3, LR-3, RF-9, XC-2, and LR-2 was increased after 72 h of germination. In addition, the AA content of XC-2 was significantly higher than other cultivars. The enhancement of essential amino acid contents was observed in a previous study of germinated rice (Kamjijam et al., 2021), which is consistent with the results in this study. Combined with the results of protein content, we conclude that part of macromolecular protein is converted into amino acids during germination (Guo et al., 2011). In addition, the 27% decrease in AA in LR-1 may be caused by new protein synthesis in the late period of seed germination. Furthermore, during germination, GAD was activated, resulting in the conversion of Glu to GABA (Komatsuzaki et al., 2007). Thus, the change in glutamic acid content plays an important role in the production of GABA. Among the seven tested cultivates, YH-3 and XC-2 had the highest content of Glu, which were 20.9% and 20.8%, respectively. After germination, the proportion of Glu in pumpkin seeds decreased slightly. We speculate that Glu is converted into GABA under the action of GAD or participated in some biochemical reactions such as protein synthesis during seed germination.
Correlation analysis (Table 2) was conducted on parameters.
GP was positively associated with sprout length (r = .941) and AA (r = .739), indicating that cultivar with higher TP is more able to ger-

| Response surface analysis of GABA germination
The results of Plackett-Burman design and significant analysis are shown in Tables S4 and S5. Among all the eight factors, germination temperature, MSG concentration, and germination time had significant effects on GABA content of XC-2, then we finally determined the optimum parameters of germination conditions by CCD (Table S6). The design model was found to be consistent with the second-order polynomial model through multiple regression analysis. The relationship between GABA content and germination parameters was shown in the following equation: where Y 1 is GABA content (mg/100 g), and A 1 , B 1 , and C 1 represent soaking temperature, MSG concentration, and germination time, The statistical analysis is listed in Table S6. To assess how well a model explains and predicts future outcomes, the coefficient of determination (R 2 ) is calculated as the proportion of the variance in the dependent variable that is predictable from the independent variables (Badwaik et al., 2012). Larger F-value and smaller p-value represent a more significant effect on the response variable, besides, if the lack of fit value was found to be not significant (p > .05), it implies that the model fits well (Quanhong & Caili, 2005). R 2 1 value of 0.88 F I G U R E 1 Variation in germination percentage, sprout length, and GABA content in pumpkin seeds before and after 72 h germination was obtained, which shows the relationship among the GABA con- When germination temperature was constant, MSG addition resulted in gradual decline in GABA content. However, the result did not show a significant (p > .05) interaction between the two independent variables (Table S6). Supplementation with glutamate can improve GABA accumulation, nevertheless, the decline in GABA content in grain with increasing glutamate concentration was reported owing to the increased enzyme-substrate complex concentration and decreasing GABA conversion rate (Iimure et al., 2009). The effects of germination temperature and germination time on GABA content are revealed in Figure 2b. Germination time had a very significant linear and quadratic effects (p < .01) on GABA accumulation. When germination temperature was set, the GABA content increased with germination time and declined later. But, the two independent variables did not interact significantly (p > .05) during the experiment. Soaking in water stimulates the enzymes activity and GABA accumulation, but too long a duration may decrease the GABA content attributed to the growth of seedling TA B L E 1 Changes in protein and amino acid content in pumpkin seeds before and after germination

TA B L E 2 Correlation analysis
between physiological indexes and main components of pumpkin seeds F I G U R E 2 Response surface plots of germination parameters (Chungcharoen et al., 2014). The effects of MSG concentration and germination time on GABA content are shown in Figure 2c. The GABA content increased with germination time. The highest point occurred in 61.6 h, then it decreased. And the interaction between MSG concentration and germination time was significant (p < .05).
By means of the RSM test results, the optimum germination parameters for GABA accumulation were soaking at 28°C for 6 h, CaCl 2 concentration of 0.2%, MSG concentration of 3.8 mg/ml, vitamin B6 concentration of 4.0 mg/ml, and germination at 30°C during 61.6 h with pH 5.8. The predicted value of GABA content was 2336 mg/100 g. Under optimized conditions, the model verification was performed. The actual content of GABA was 2319 ± 10 mg/100 g and the relative error was only 0.7% when compared with predicted value. The results above indicated that the model was valid; besides, the combination of Plackett-Burman design and CCD method was reliable. After germination, the GABA content was 5.4 times higher than that of the nongerminated XC-2.

| Response surface analysis of UAE
Based on the result of single-factor experiments, Box-Behnken design was performed to optimize the parameters of UAE conditions, including solid-liquid ratio, ultrasonic power, time, and temperature.
According to the result of multiple regression analysis, the design model was found to be consistent with the second-order polynomial model. The relationship between GABA yield and UAE parameters is shown in the following equation: where Y 2 is GABA yield (mg/100 g), and A 2 , B 2 , C 2 , and D 2 are solidliquid ratio, ultrasonic power, time, and temperature, respectively.
The statistical analysis is listed in Table S7. The model was significant with a satisfactory value of R 2 2 (R 2 2 = 0.92). An F-value of 12.03 and p < .0001 indicated the model was highly significant, besides, the lack of fit value was found to be not significant (p = .20), which proved the validity of the model. The ANOVA results showed that solid-liquid ratio has a highly significant (p < .01) effect on GABA yield.
Meanwhile, GABA yield was significantly affected by ultrasonic time and temperature. Additionally, interaction between solid-liquid ratio and ultrasonic temperature showed highly significant effect on GABA yield, ultrasonic power, and time interacted in the same way.
Response surfaces plots (Figure 3) were also applied to illustrate the interaction of solid-liquid ratio, ultrasonic power, time, and temperature on GABA extraction efficiency. Effects of solid-liquid ratio and ultrasonic power on GABA extraction are revealed in Figure 3a.
Solid-liquid ratio had a very significant (p < .01) effect on GABA yield at linear and quadratic level. When ultrasonic power was constant, GABA yield was sharply increased and then descended with the raise in solid-liquid ratio. However, the interaction between solid-liquid ratio and ultrasonic power was not significant (p > .05) ( Table S7).
The effects of solid-liquid ratio and ultrasonic time during UAE treatment are presented in Figure 3b. The two independent variables had quadratic effects on GABA yield during extraction. According to the variation trend, the effect of solid-liquid ratio contributed more to GABA yield. Also, no significant (p > .05) interaction was observed between the two independent variables. Figure 3c illustrates the effects of ultrasonic power and temperature during extraction. The increased the solubility of GABA. However, the dissolution of other component in pumpkin seeds also increased when the system was overheated (Pinelo et al., 2005). The effect of ultrasonic temperature at linear lever is revealed in Figure 3e,

| Hypolipidemic effect of PSGE in T2DM rats
T2DM rat model was established by HFD feeding and injection of STZ. Then, two groups of T2DM rats were treated with PSGE and MT, respectively. After 4 weeks, biochemical parameters of serum were determined and the results are listed in Table 3. The level of TC, TG, and LDL was significantly (p < .05) reduced after treating with PSGE as compared with the T2DM group. Moreover, the level of LDL in PSGE group was significantly lower than that in the MT group. The results above were consistent with a study in which the regulation effects of GABA on lipid levels in serum were reported (Rashmi et al., 2018;Sato et al., 2021). Besides, it is demonstrated that germinated brown rice could reduce the level of TC, TG, and LDL (Hsu et al., 2008;Roohinejad et al., 2010), which may relate to GABA accumulation during germination experiments on animals.
Several studies reported that insulin sensitivity and glucose toler-

| CON CLUS ION
The combined technique of germination and UAE was effective for the enhancement of GABA yield in pumpkin seed. The germination parameters and UAE parameters were successfully screened by BBD and CCD of RSM. The optimal germinated conditions were as follows: soaking the seeds at 28°C for 6 h with 0.2% CaCl 2 , 3.8 mg/ ml monosodium glutamate, and 4.0 mg/ml vitamin B 6 , then germination was carried out at 30°C for 61.6 h. After germination with the optimal conditions, the GABA content of XC-2 was 5.40 times higher than that of raw material. The optimal UAE conditions were Note: Data are presented as mean ± SD. Values in the same row with different letters (a-c) are significantly different, p < .05.

TA B L E 3
The effects of PSGE on serum biochemical parameters of T2DM rats Furthermore, the regulation of TC, TG, and HDL was observed in T2DM rats after being treated with PSGE, which demonstrated the potential hypolipidemic effect of GABA extract from the pumpkin seed. In conclusion, the application of germination and UAE efficiently provided a GABA-enriched production of pumpkin seed, which could contribute to health benefits related to lifestyleassociated diseases.

ACK N OWLED G M ENTS
This work was supported by the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910011025) and the National Natural Science Foundation of China (Nos. 32122069 and 31972191).

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.